Micronized placental tissue compositions and methods of making and using the same

ABSTRACT

Described herein are compositions composed of micronized placental components and pharmaceutical compositions thereof. The compositions have numerous medical applications. Methods for making and using the micronized compositions are also described herein.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority upon U.S. provisional application Ser.No. 61/442,346, filed Feb. 14, 2011, and Ser. No. 61/543,995, filed Oct.6, 2011. These applications are hereby incorporated by reference intheir entireties for all of their teachings.

BACKGROUND

Placental tissue is typically harvested after an elective Cesareansurgery. The placenta is composed of the umbilical cord and amnioticsac. The amniotic sac, commonly referred to as the amniotic membrane,has two primary layers of tissue, amnion and chorion. Amnion tissue isthe innermost layer of the amniotic sac and in direct contact with theamniotic fluid. The amniotic sac contains the amniotic fluid andprotects the fetal environment. Histological evaluation indicates thatthe membrane layers of the amnion consist of single layer of epitheliumcells, thin reticular fibers (basement membrane), a thick compact layer,and fibroblast layer. The fibrous layer of amnion (i.e., the basementmembrane) contains collagen types IV, V, and VII, and cell-adhesionbio-active factors including fibronectin and laminins. Described hereinis unique approach to using placental tissue components in wound healingand other medical applications.

SUMMARY

Described herein are composition composed of micronized placentalcomponents and pharmaceutical compositions thereof. The compositionshave numerous medical applications. Methods for making and using themicronized compositions are also described herein.

The advantages of the invention will be set forth in part in thedescription which follows, and in part will be obvious from thedescription, or may be learned by practice of the aspects describedbelow. The advantages described below will be realized and attained bymeans of the elements and combinations particularly pointed out in theappended claims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several aspects described below.

FIG. 1 is an overview flow chart of the process for making themicronized compositions described herein.

DETAILED DESCRIPTION

Before the present articles and methods are disclosed and described, itis to be understood that the aspects described below are not limited tospecific compounds, synthetic methods, or uses as such may, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular aspects only and is notintended to be limiting.

In this specification and in the claims that follow, reference will bemade to a number of terms that shall be defined to have the followingmeanings:

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise. Thus, for example,reference to “a bioactive agent” includes mixtures of two or more suchagents, and the like.

“Optional” or “optionally” means that the subsequently described eventor circumstance can or cannot occur, and that the description includesinstances where the event or circumstance occurs and instances where itdoes not. For example, the phrase “optionally cleaning step” means thatthe cleaning step may or may not be performed.

The term “subject” as used herein is any vertebrate organism.

The term “amnion” as used herein includes amniotic membrane where theintermediate tissue layer is intact or has been substantially removed.

Titles or subtitles may be used in the specification for the convenienceof a reader, which are not intended to influence the scope of thepresent invention. Additionally, some terms used in this specificationare more specifically defined below.

I. Micronized Compositions and Methods for Making Thereof

Described herein are compositions composed of micronized placentalcomponents and pharmaceutical compositions thereof. In one aspect, thecomposition includes (a) micronized amnion, chorion, intermediate tissuelayer, or any combination thereof and (b) a pharmaceutically acceptablecarrier. In one aspect, the composition includes micronized amnion andintermediate tissue layer. In another aspect, the composition includesmicronized amnion and chorion.

FIG. 1 depicts an overview (100) and certain aspects of the steps toharvest, process, and prepare placental material for use in thepreparation of the micronized compositions described herein. Moredetailed descriptions and discussion regarding each individual step willfollow. Initially, the placental tissue is collected from a consentingpatient following an elective Cesarean surgery (step 110). The materialis preserved and transported in conventional tissue preservation mannerto a suitable processing location or facility for check-in andevaluation (step 120). Gross processing, handling, and separation of thetissue layers then takes place (step 130). Acceptable tissue is thendecontaminated (step 140) and dehydrated (step 145). Afterdecontamination dehydration, the placental components (e.g., amnion,intermediate tissue layer and/or chorion individually or as grafts) arethen micronized (step 150). Each step is described in detail below.

Initial Tissue Collection (Step 110)

The components used to produce the micronized compositions describedherein are derived from the placenta. The source of the placenta canvary. In one aspect, the placenta is derived from a mammal such as humanand other animals including, but not limited to, cows, pigs, and thelike can be used herein. In the case of humans, the recovery of theplacenta originates in a hospital, where it is collected during aCesarean section birth. The donor, referring to the mother who is aboutto give birth, voluntarily submits to a comprehensive screening processdesigned to provide the safest tissue possible for transplantation. Thescreening process preferably tests for antibodies to the humanimmunodeficiency virus type 1 and type 2 (anti-HIV-1 and anti-HIV-2),antibodies to the hepatitis B virus (anti-HBV) hepatitis B surfaceantigens (HBsAg), antibodies to the hepatitis C virus (anti-HCV),antibodies to the human T-lymphotropic virus type I and type II(anti-HTLV-I, anti-HTLV-II), CMV, and syphilis, and nucleic acid testingfor human immune-deficiency virus type 1 (HIV-1) and for the hepatitis Cvirus (HCV), using conventional serological tests. The above list oftests is exemplary only, as more, fewer, or different tests may bedesired or necessary over time or based upon the intended use of thegrafts, as will be appreciated by those skilled in the art.

Based upon a review of the donor's information and screening testresults, the donor will either be deemed acceptable or not. In addition,at the time of delivery, cultures are taken to determine the presence ofbacteria, for example, Clostridium or Streptococcus. If the donor'sinformation, screening tests, and the delivery cultures are allsatisfactory (i.e., do not indicate any risks or indicate acceptablelevel of risk), the donor is approved by a medical director and thetissue specimen is designated as initially eligible for furtherprocessing and evaluation.

Human placentas that meet the above selection criteria are preferablybagged in a saline solution in a sterile shipment bag and stored in acontainer of wet ice for shipment to a processing location or laboratoryfor further processing.

If the placenta is collected prior to the completion of obtaining theresults from the screening tests and delivery cultures, such tissue islabeled and kept in quarantine. The placenta is approved for furtherprocessing only after the required screening assessments and deliverycultures, which declare the tissue safe for handling and use, aresatisfied and obtains final approval from a medical director.

Material Check-in and Evaluation (Step 120)

Upon arrival at the processing center or laboratory, the shipment isopened and verified that the sterile shipment bag/container is stillsealed and in the coolant, that the appropriate donor paperwork ispresent, and that the donor number on the paperwork matches the numberon the sterile shipment bag containing the tissue. The sterile shipmentbag containing the tissue is then stored in a refrigerator until readyfor further processing.

Gross Tissue Processing (Step 130)

When the tissue is ready to be processed further, the sterile suppliesnecessary for processing the placental tissue further are assembled in astaging area in a controlled environment and are prepared forintroduction into a controlled environment. In one aspect, the placentais processed at room temperature. If the controlled environment is amanufacturing hood, the sterile supplies are opened and placed into thehood using conventional sterilization techniques. If the controlledenvironment is a clean room, the sterile supplies are opened and placedon a cart covered by a sterile drape. All the work surfaces are coveredby a piece of sterile drape using conventional sterilization techniques,and the sterile supplies and the processing equipment are placed ontothe sterile drape, again using conventional sterilization techniques.

Processing equipment is decontaminated according to conventional andindustry-approved decontamination procedures and then introduced intothe controlled environment. The equipment is strategically placed withinthe controlled environment to minimize the chance for the equipment tocome in proximity to or is inadvertently contaminated by the tissuespecimen.

Next, the placenta is removed from the sterile shipment bag andtransferred aseptically to a sterile processing basin within thecontrolled environment. The sterile basin contains hyperisotonic salinesolution (e.g., 18% NaCl) that is at room or near room temperature. Theplacenta is gently massaged to help separate blood clots and to allowthe placental tissue to reach room temperature, which facilitates theseparation of the placental components from each other (e.g., amnionmembrane and chorion). After having warmed up to ambient temperature(e.g., after about 10-30 minutes), the placenta is then removed from thesterile processing basin and laid flat on a processing tray with theamnion membrane layer facing down for inspection.

The placenta is examined for discoloration, debris or othercontamination, odor, and signs of damage. The size of the tissue is alsonoted. A determination is made, at this point, as to whether the tissueis acceptable for further processing.

The amnion and chorion are next carefully separated. In one aspect, thematerials and equipment used in this procedure include a processingtray, 18% saline solution, sterile 4×4 sponges, and two sterile Nalgenejars. The placenta tissue is then closely examined to find an area(typically a corner) in which the amnion can be separated from thechorion. The amnion appears as a thin, opaque layer on the chorion.

The fibroblast layer is identified by gently contacting each side of theamnion with a piece of sterile gauze or a cotton tipped applicator. Thefibroblast layer will stick to the test material. The amnion is placedinto processing tray basement membrane layer down. Using a bluntinstrument, a cell scraper, or sterile gauze, any residual blood is alsoremoved. This step must be done with adequate care, again, so as not totear the amnion. The cleaning of the amnion is complete once the amnionis smooth and opaque-white in appearance.

In certain aspects, the intermediate tissue layer, also referred to asthe spongy layer, is substantially removed from the amnion in order toexpose the fibroblast layer. The term “substantially removed” withrespect to the amount of intermediate tissue layer removed is definedherein as removing greater than 90%, greater than 95%, or greater than99% of the intermediate tissue layer from the amnion. This can beperformed by peeling the intermediate tissue layer from the amnion.Alternatively, the intermediate tissue layer can be removed from theamnion by wiping the intermediate tissue layer with gauze or othersuitable wipe. The resulting amnion can be subsequently decontaminatedusing the process described below.

In certain aspects, the epithelium layer present on the amnion issubstantially removed in order to expose the basement layer of theamnion. The term “substantially removed” with respect to the amount ofepithelium removed is defined herein as removing greater than 90%,greater than 95%, or greater than 99% of the epithelial cells from theamnion. The presence or absence of epithelial cells remaining on theamnion layer can be evaluated using techniques known in the art. Forexample, after removal of the epithelial cell layer, a representativetissue sample from the processing lot is placed onto a standardmicroscope examination slide. The tissue sample is then stained usingEosin Y Stain and evaluated as described below. The sample is thencovered and allowed to stand. Once an adequate amount of time has passedto allow for staining, visual observation is done under magnification.

The epithelium layer can be removed by techniques known in the art. Forexample, the epithelium layer can be scraped off of the amnion using acell scraper. Other techniques include, but are not limited to, freezingthe membrane, physical removal using a cell scraper, or exposing theepithelial cells to nonionic detergents, anionic detergents, andnucleases. The de-epithelialized tissue is then evaluated to determinethat the basement membrane has not been compromised and remains intact.This step is performed after completion of the processing step and thebefore the tissue has been dehydrated as described in the next section.For example, a representative sample graft is removed for microscopicanalysis. The tissue sample is place onto a standard slide, stained withEosin Y and viewed under the microscope. If epithelium is present, itwill appear as cobblestone-shaped cells.

The methods described herein do not remove all cellular components inthe amnion. This technique is referred to in the art as“decellularization.” Decellularization generally involves the physicaland/or chemical removal of all cells present in the amnion, whichincludes epithelial cells and fibroblast cells. For example, althoughthe removal of epithelial cells is optional, the fibroblast layerpresent in the amnion stromal layer is intact, even if the intermediatetissue layer is removed.

Chemical Decontamination (Step 140)

The amnion and chorion isolated above can be chemically decontaminatedusing the techniques described below. In one aspect, the amnion andchorion is decontaminated at room temperature. In one aspect, the amnionproduced in step 130 (e.g., with or without the intermediate tissuelayer) can be placed into a sterile Nalgene jar for the next step. Inone aspect, the following procedure can be used to clean the amnion. ANalgene jar is aseptically filled with 18% saline hypertonic solutionand sealed (or sealed with a top). The jar is then placed on a rockerplatform and agitated for between 30 and 90 minutes, which furthercleans the amnion of contaminants. If the rocker platform was not in thecritical environment (e.g., the manufacturing hood), the Nalgene jar isreturned to the controlled/sterile environment and opened. Using sterileforceps or by aseptically decanting the contents, the amnion is gentlyremoved from the Nalgene jar containing the 18% hyperisotonic salinesolution and placed into an empty Nalgene jar. This empty Nalgene jarwith the amnion is then aseptically filled with a premixed antibioticsolution. In one aspect, the premixed antibiotic solution is composed ofa cocktail of antibiotics, such as Streptomycin Sulfate and GentamicinSulfate. Other antibiotics, such as Polymixin B Sulfate and Bacitracin,or similar antibiotics now available or available in the future, arealso suitable. Additionally, it is preferred that the antibioticsolution be at room temperature when added so that it does not changethe temperature of or otherwise damage the amnion. This jar or containercontaining the amnion and antibiotics is then sealed or closed andplaced on a rocker platform and agitated for, preferably, between 60 and90 minutes. Such rocking or agitation of the amnion within theantibiotic solution further cleans the tissue of contaminants andbacteria. Optionally, the amnion can be washed with a detergent. In oneaspect, the amnion can be washed with 0.1 to 10%, 0.1 to 5%, 0.1 to 1%,or 0.5% Triton-X wash solution.

If the rocker platform was not in the critical environment (e.g., themanufacturing hood), the jar or container containing the amnion andantibiotics is then returned to the critical/sterile environment andopened. Using sterile forceps, the amnion is gently removed from the jaror container and placed in a sterile basin containing sterile water ornormal saline (0.9% saline solution). The amnion is allowed to soak inplace in the sterile water/normal saline solution for at least 10 to 15minutes. The amnion may be slightly agitated to facilitate removal ofthe antibiotic solution and any other contaminants from the tissue.After at least 10 to 15 minutes, the amnion is ready to be dehydratedand processed further.

In the case of chorion, the following exemplary procedure can be used.After separation of the chorion from the amnion and removal of clottedblood from the fibrous layer, the chorion is rinsed in 18% salinesolution for 15 minutes to 60 minutes. During the first rinse cycle, 18%saline is heated in a sterile container using a laboratory heating platesuch that the solution temperature is approximately 48° C. The solutionis decanted, the chorion tissue is placed into the sterile container,and decanted saline solution is poured into the container. The containeris sealed and placed on a rocker plate and agitated for 15 minutes to 60minutes. After 1 hour agitation bath, the chorion tissue was removed andplaced into second heated agitation bath for an additional 15 minutes to60 minutes rinse cycle. Optionally, the chorion tissue can be washedwith a detergent (e.g., Triton-X wash solution) as discussed above forthe decontamination of amnion. The container is sealed and agitatedwithout heat for 15 minutes to 120 minutes. The chorion tissue is nextwashed with deionized water (250 ml of DI water×4) with vigorous motionfor each rinse. The tissue is removed and placed into a container of1×PBS w/EDTA solution. The container is sealed and agitated for 1 hourat controlled temperature for 8 hours. The chorion tissue is removed andrinsed using sterile water. A visual inspection was performed to removeany remaining discolored fibrous blood material from the chorion tissue.The chorion tissue should have a cream white visual appearance with noevidence of brownish discoloration.

Dehydration (Step 145)

In one aspect, the amnion, chorion, intermediate tissue layer, or anycombination thereof can be processed into tissue grafts (i.e.,laminates) that are subsequently micronized. In another aspect, theindividual amnion, chorion, intermediate tissue layers can be dehydratedindependently and subsequently micronized alone or a s a mixture ofcomponents. In one aspect, the tissue (i.e., individual membrane orgraft) is dehydrated by chemical dehydration followed by freeze-drying.In one aspect, the chemical dehydration step is performed by contactingthe amnion, chorion, and/or intermediate layer with a polar organicsolvent for a sufficient time and amount in order to substantially(i.e., greater than 90%, greater than 95%, or greater than 99%) orcompletely remove residual water present in the tissue (i.e., dehydratethe tissue). The solvent can be protic or aprotic. Examples of polarorganic solvents useful herein include, but are not limited to,alcohols, ketones, ethers, aldehydes, or any combination thereof.Specific, non-limiting examples include DMSO, acetone, tetrahydrofuran,ethanol, isopropanol, or any combination thereof. In one aspect, theplacental tissue is contacted with a polar organic solvent at roomtemperature. No additional steps are required, and the tissue can befreeze-dried directly as discussed below.

After chemical dehydration, the tissue is freeze-dried in order toremove any residual water and polar organic solvent. In one aspect, theamnion, chorion, and/or intermediate layer can be laid on a suitabledrying fixture prior to freeze-drying. For example, one or more stripsof amnion can be laid on a suitable drying fixture. Next, chorion islaid on top of the amnion. In this aspect, an amnion/chorion tissuegraft is produced. Alternatively, a strip of amnion can be placed on afirst drying fixture, and a strip of chorion can be placed on a seconddrying fixture. The drying fixture is preferably sized to be largeenough to receive the placental tissue, fully, in laid out, flatfashion. In one aspect, the drying fixture is made of Teflon or ofDelrin, which is the brand name for an acetal resin engineering plasticinvented and sold by DuPont and which is also available commerciallyfrom Werner Machine, Inc. in Marietta, Ga. Any other suitable materialthat is heat and cut resistant, capable of being formed into anappropriate shape to receive wet tissue can also be used for the dryingfixture.

Once the tissue is placed on the drying fixture, the drying fixture isplaced in the freeze-dryer. The use of the freeze-dryer to dehydrate thetissue can be more efficient and thorough compared to other techniquessuch as thermal dehydration. In general, it is desirable to avoid icecrystal formation in the placental tissue as this may damage theextracellular matrix in the tissue. By chemically dehydrating theplacental tissue prior to freeze-drying, this problem can be avoided.

In another aspect, the dehydration step involves applying heat to thetissue. In one aspect, the amnion, chorion, and/or intermediate layer islaid on a suitable drying fixture (either as individual strips or as alaminate discussed above), and the drying fixture is placed in a sterileTyvex (or similar, breathable, heat-resistant, and sealable material)dehydration bag and sealed. The breathable dehydration bag prevents thetissue from drying too quickly. If multiple drying fixtures are beingprocessed simultaneously, each drying fixture is either placed in itsown Tyvex bag or, alternatively, placed into a suitable mounting framethat is designed to hold multiple drying frames thereon and the entireframe is then placed into a larger, single sterile Tyvex dehydration bagand sealed.

The Tyvex dehydration bag containing the one or more drying fixtures isthen placed into a non-vacuum oven or incubator that has been preheatedto approximately 35 to 50 degrees Celcius. The Tyvex bag remains in theoven for between 30 to 120 minutes. In one aspect, the heating step canbe performed at 45 minutes at a temperature of approximately 45 degreesCelcius to dry the tissue sufficiently but without over-drying orburning the tissue. The specific temperature and time for any specificoven will need to be calibrated and adjusted based on other factorsincluding altitude, size of the oven, accuracy of the oven temperature,material used for the drying fixture, number of drying fixtures beingdried simultaneously, whether a single or multiple frames of dryingfixtures are dried simultaneously, and the like.

Preparation of Micronized Compositions and Pharmaceutical CompositionsThereof (Step 150)

Once the amnion, chorion, and/or intermediate tissue layer have beendehydrated individually or in the form a of tissue graft, the dehydratedtissue(s) is micronized. The micronized compositions can be producedusing instruments known in the art. For example, the Retsch OscillatingMill MM400 can be used to produce the micronized compositions describedherein. The particle size of the materials in the micronized compositioncan vary as well depending upon the application of the micronizedcomposition. In one aspect, the micronized composition has particlesthat are less than 500 μm, less than 400 μm, less than 300 μm, or from25 μm to 300 μm, from 25 μm to 200 μm, or from 25 μm to 150 μm. Incertain aspects, particles having a larger diameter (e.g. 150 μm to 350μm) are desirable.

In one aspect, micronization is performed by mechanical grinding orshredding. In another aspect, micronization is performed cryogenicgrinding. In this aspect, the grinding jar containing the tissue iscontinually cooled with liquid nitrogen from the integrated coolingsystem before and during the grinding process. Thus the sample isembrittled and volatile components are preserved. Moreover, thedenaturing of proteins in the amnion, intermediate tissue layer, and/orchorion is minimized or prevented. In one aspect, the CryoMillmanufactured by Retsch can be used in this aspect.

The selection of components used to make the micronized componentsdescribed herein can vary depending upon the end-use of the composition.For example, amnion, chorion, intermediate tissue layer, or anycombination thereof as individual components can be admixed with oneanother and subsequently micronized. In another aspect, one or moretissue grafts composed of one or more amnion, chorion, intermediatetissue layers, or any combination thereof (i.e., laminates) can bemicronized. In a further aspect, one or more tissue grafts composed ofone or more amnion, chorion, intermediate tissue layers, or anycombination can be admixed with amnion, chorion, intermediate tissuelayer, or any combination thereof as individual components andsubsequently micronized.

The amount of different components used to make the micronizedcompositions described herein can vary depending upon the application ofthe micronized composition. In one aspect, when the micronizedcomposition is composed of amnion (with or without the intermediatetissue layer) and intermediate tissue layer, the weight ratio of amnionto intermediate tissue layer is from 10:1 to 1:10, 9:1 to 1:1, 8:1 to1:1, 7:1 to 1:1, 6:1 to 1:1, 5:1 to 1:1, 4:1 to 1:1, 3:1 to 1:1, 2:1 to1:1, or about 1:1. In another aspect, when the micronized composition iscomposed of amnion (with or without the intermediate tissue layer) andchorion, the weight ratio of chorion to amnion is from 10:1 to 1:10, 9:1to 1:1, 8:1 to 1:1, 7:1 to 1:1, 6:1 to 1:1, 5:1 to 1:1, 4:1 to 1:1, 3:1to 1:1, 2:1 to 1:1, or about 1:1.

In addition to amnion, the intermediate tissue layer, and chorion,additional components can be added to the composition prior to and/orafter micronization. In one aspect, a filler can be added. Examples offillers include, but are not limited to, allograft pericardium,allograft acellular dermis, Wharton's jelly separated from vascularstructures (i.e., umbilical vein and artery) and surrounding membrane,purified xenograft Type-1 collagen, biocellulose polymers or copolymers,biocompatible synthetic polymer or copolymer films, purified smallintestinal submucosa, bladder acellular matrix, cadaveric fascia, or anycombination thereof.

In another aspect, a bioactive agent can be added to the compositionprior to and/or after micronization. Examples of bioactive agentsinclude, but are not limited to, naturally occurring growth factorssourced from platelet concentrates, either using autologous bloodcollection and separation products, or platelet concentrates sourcedfrom expired banked blood; bone marrow aspirate; stem cells derived fromconcentrated human placental cord blood stem cells, concentratedamniotic fluid stem cells or stem cells grown in a bioreactor; orantibiotics. Upon application of the micronized composition withbioactive agent to the region of interest, the bioactive agent isdelivered to the region over time. Thus, the micronized particlesdescribed herein are useful as delivery devices of bioactive agents andother pharmaceutical agents when administered to a subject. Releaseprofiles can be modified based on, among other things, the selection ofthe components used to make the micronized compositions as well as thesize of the particles.

In a further aspect, the amnion can be cross-linked with theintermediate tissue layer, chorion, or a second amnion tissue. Forexample, a cross-linking agent can be added to the composition (e.g.,amnion, chorion, intermediate tissue layer, or any combination thereofas individual components and/or as tissue grafts) prior to and/or aftermicronization. In general, the cross-linking agent is nontoxic andnon-immunogenic. When the amnion, intermediate tissue layer, and/orchorion (or a tissue graft thereof) are treated with the cross-linkingagent, the cross-linking agent can be the same or different. In oneaspect, the amnion, intermediate tissue layer, and chorion can betreated separately with a cross-linking agent or, in the alternative,the amnion, intermediate tissue layer, and chorion can be treatedtogether with the same cross-linking agent. In certain aspects, theamnion, intermediate tissue layer, and chorion can be treated with twoor more different cross-linking agents. The conditions for treating theamnion, intermediate tissue layer, and chorion can vary. In otheraspects, the amnion, intermediate tissue layer, and/or chorion can bemicronized, and the micronized composition can subsequently be treatedwith a cross-linking agent. In one aspect, the concentration of thecross-linking agent is from 0.1 M to 5 M, 0.1 M to 4 M, 0.1 M to 3 M,0.1 M to 2 M, or 0.1 M to 1 M.

The cross-linking agent generally possesses two or more functionalgroups capable of reacting with proteins to produce covalent bonds. Inone aspect, the cross-linking agent possesses groups that can react withamino groups present on the protein. Examples of such functional groupsinclude, but are not limited to, hydroxyl groups, substituted orunsubstituted amino groups, carboxyl groups, and aldehyde groups. In oneaspect, the cross-linker can be a dialdehyde such as, for example,glutaraldehyde. In another aspect, the cross-linker can be acarbodiimide such as, for example,(N-(3-dimethylaminopropyl)-N′-ethyl-carbodiimide (EDC). In otheraspects, the cross-linker can be an oxidized dextran, p-azidobenzoylhydrazide, N-[alpha-maleimidoacetoxy]succinimide ester, p-azidophenylglyoxal monohydrate, bis-[beta-(4-azidosalicylamido)ethyl]disulfide,bis-[sulfosuccinimidyl]suberate, dithiobis[succinimidyl]propionate,disuccinimidyl suberate, and1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride, abifunctional oxirane (OXR), or ethylene glycol diglycidyl ether (EGDE).

In one aspect, sugar is the cross-linking agent, where the sugar canreact with proteins present in the amnion, intermediate tissue layer,and chorion to form a covalent bond. For example, the sugar can reactwith proteins by the Maillard reaction, which is initiated by thenonenzymatic glycosylation of amino groups on proteins by reducingsugars and leads to the subsequent formation of covalent bonds. Examplesof sugars useful as a cross-linking agent include, but are not limitedto, D-ribose, glycerose, altrose, talose, ertheose, glucose, lyxose,mannose, xylose, gulose, arabinose, idose, allose, galactose, maltose,lactose, sucrose, cellibiose, gentibiose, melibiose, turanose,trehalose, isomaltose, or any combination thereof.

In certain aspects, the micronized composition can be used to form athree-dimensional construct. For example, the micronized particles canbe treated with a cross-linking agent described above then placed in amold having specific dimensions. Alternatively, the micronized particlescan be placed into the mold and subsequently treated with thecross-linking agent. In one aspect, the cross-linked particles can bemanually formed into any desired shape. In other aspects, one or moreadhesives can be admixed with an adhesive prior to being introduced intothe mold. Examples of such adhesives include, but are not limited to,fibrin sealants, cyanoacrylates, gelatin and thrombin products,polyethylene glycol polymer, albumin, and glutaraldehyde products. Notwishing to be bound by theory, the three-dimensional construct composedof smaller micronized particles will produce a denser product capable ofbearing mechanical loads. Alternatively, larger micronized particleswill produce constructs that are less dense and possess compressiveproperties. This feature can be useful in non-load void filling,especially where it is desirable to have a product that will conform toirregular shapes. The three-dimensional constructs can include one ormore bioactive agents described herein.

In other aspects, the micronized compositions described herein can beformulated in any excipient the biological system or entity can tolerateto produce pharmaceutical compositions. Examples of such excipientsinclude, but are not limited to, water, aqueous hyaluronic acid, saline,Ringer's solution, dextrose solution, Hank's solution, and other aqueousphysiologically balanced salt solutions. Nonaqueous vehicles, such asfixed oils, vegetable oils such as olive oil and sesame oil,triglycerides, propylene glycol, polyethylene glycol, and injectableorganic esters such as ethyl oleate can also be used. Other usefulformulations include suspensions containing viscosity enhancing agents,such as sodium carboxymethylcellulose, sorbitol, or dextran. Excipientscan also contain minor amounts of additives, such as substances thatenhance isotonicity and chemical stability. Examples of buffers includephosphate buffer, bicarbonate buffer and Tris buffer, while examples ofpreservatives include thimerosol, cresols, formalin and benzyl alcohol.In certain aspects, the pH can be modified depending upon the mode ofadministration. Additionally, the pharmaceutical compositions caninclude carriers, thickeners, diluents, preservatives, surface activeagents and the like in addition to the compounds described herein.

The pharmaceutical compositions can be prepared using techniques knownin the art. In one aspect, the composition is prepared by admixing amicronized composition described herein with apharmaceutically-acceptable compound and/or carrier. The term “admixing”is defined as mixing the two components together so that there is nochemical reaction or physical interaction. The term “admixing” alsoincludes the chemical reaction or physical interaction between thecompound and the pharmaceutically-acceptable compound.

It will be appreciated that the actual preferred amounts of micronizedcomposition in a specified case will vary according to the specificcompound being utilized, the particular compositions formulated, themode of application, and the particular situs and subject being treated.Dosages for a given host can be determined using conventionalconsiderations, e.g. by customary comparison of the differentialactivities of the subject compounds and of a known agent, e.g., by meansof an appropriate conventional pharmacological protocol. Physicians andformulators, skilled in the art of determining doses of pharmaceuticalcompounds, will have no problems determining dose according to standardrecommendations (Physician's Desk Reference, Barnhart Publishing (1999).

The pharmaceutical compositions described herein can be administered ina number of ways depending on whether local or systemic treatment isdesired, and on the area to be treated. In one aspect, administrationcan be by injection, where the micronized composition is formulated intoa liquid or gel. In other aspects, the micronized composition can beformulated to be applied internally to a subject. In other aspects, themicronized composition can be applied topically (includingophthalmically, vaginally, rectally, intranasally, orally, or directlyto the skin).

In one aspect, the micronized compositions can be formulated as atopical composition applied directly to the skin. Formulations fortopical administration can include, emulsions, creams, aqueoussolutions, oils, ointments, pastes, gels, lotions, milks, foams,suspensions and powders. In one aspect, the topical composition caninclude one or more surfactants and/or emulsifiers.

Surfactants (or surface-active substances) that may be present areanionic, non-ionic, cationic and/or amphoteric surfactants. Typicalexamples of anionic surfactants include, but are not limited to, soaps,alkylbenzenesulfonates, alkanesulfonates, olefin sulfonates, alkyl ethersulfonates, glycerol ether sulfonates, .alpha.-methyl ester sulfonates,sulfo fatty acids, alkyl sulphates, fatty alcohol ether sulphates,glycerol ether sulphates, fatty acid ether sulphates, hydroxy mixedether sulphates, monoglyceride (ether) sulphates, fatty acid amide(ether) sulphates, mono- and dialkyl sulfosuccinates, mono- and dialkylsulfosuccinamates, sulfotriglycerides, amide soaps, ether carboxylicacids and salts thereof, fatty acid isethionates, fatty acidsarcosinates, fatty acid taurides, N-acylamino acids, e.g. acyllactylates, acyl tartrates, acyl glutamates and acyl aspartates, alkyloligoglucoside sulphates, protein fatty acid condensates (in particularwheat-based vegetable products) and alkyl (ether) phosphates. Examplesof non-ionic surfactants include, but are not limited to, fatty alcoholpolyglycol ethers, alkylphenol polyglycol ethers, fatty acid polyglycolesters, fatty acid amide polyglycol ethers, fatty amine polyglycolethers, alkoxylated triglycerides, mixed ethers or mixed formals,optionally partially oxidized alk(en)yl oligoglycosides or glucoronicacid derivatives, fatty acid N-alkylglucamides, protein hydrolysates (inparticular wheat-based vegetable products), polyol fatty acid esters,sugar esters, sorbitan esters, polysorbates and amine oxides. Examplesof amphoteric or zwitterionic surfactants include, but are not limitedto, alkylbetaines, alkylamidobetaines, aminopropionates,aminoglycinates, imidazolinium-betaines and sulfobetaines.

In one aspect, the surfactant can be fatty alcohol polyglycol ethersulphates, monoglyceride sulphates, mono- and/or dialkylsulfosuccinates, fatty acid isethionates, fatty acid sarcosinates, fattyacid taurides, fatty acid glutamates, alpha-olefinsulfonates, ethercarboxylic acids, alkyl oligoglucosides, fatty acid glucamides,alkylamidobetaines, amphoacetals and/or protein fatty acid condensates.

Examples of zwitterionic surfactants include betaines, such asN-alkyl-N,N-dimethylammonium glycinates, for examplecocoalkyldimethylammonium glycinate,N-acylaminopropyl-N,N-dimethylammonium glycinates, for examplecocoacylaminopropyldimethylammonium glycinate, and2-alkyl-3-carboxymethyl-3-hydroxyethylimidazolines having in each case 8to 18 carbon atoms in the alkyl or acyl group, andcocoacylaminoethylhydroxyethyl-carboxymethyl glycinate.

In one aspect, the emulsifier can be a nonionogenic surfactant selectedfrom the following: addition products of from 2 to 30 mol of ethyleneoxide and/or 0 to 5 mol of propylene oxide onto linear fatty alcoholshaving 8 to 22 carbon atoms, onto fatty acids having 12 to 22 carbonatoms, onto alkylphenols having 8 to 15 carbon atoms in the alkyl group,and onto alkylamines having 8 to 22 carbon atoms in the alkyl radical;alkyl and/or alkenyl oligoglycosides having 8 to 22 carbon atoms in thealk(en)yl radical and the ethoxylated analogs thereof; addition productsof from 1 to 15 mol of ethylene oxide onto castor oil and/orhydrogenated castor oil; addition products of from 15 to 60 mol ofethylene oxide onto castor oil and/or hydrogenated castor oil; partialesters of glycerol and/or sorbitan with unsaturated, linear orsaturated, branched fatty acids having 12 to 22 carbon atoms and/orhydroxycarboxylic acids having 3 to 18 carbon atoms, and the adductsthereof with 1 to 30 mol of ethylene oxide; partial esters ofpolyglycerol (average degree of self-condensation 2 to 8),trimethylolpropane, pentaerythritol, sugar alcohols (e.g. sorbitol),alkyl glucosides (e.g. methyl glucoside, butyl glucoside, laurylglucoside), and polyglucosides (e.g. cellulose) with saturated and/orunsaturated, linear or branched fatty acids having 12 to 22 carbon atomsand/or hydroxycarboxylic acids having 3 to 18 carbon atoms, and theadducts thereof with 1 to 30 mol of ethylene oxide; mixed esters ofpentaerythritol, fatty acids, citric acid and fatty alcohols and/ormixed esters of fatty acids having 6 to 22 carbon atoms, methylglucoseand polyols, preferably glycerol or polyglycerol, mono-, di- andtrialkyl phosphates, and mono-, di- and/or tri-PEG alkyl phosphates andsalts thereof; wool wax alcohols; polysiloxane-polyalkyl-polyethercopolymers and corresponding derivatives; and block copolymers, e.g.polyethylene glycol-30 dipolyhydroxystearates. In one aspect, theemulsifier is a polyalkylene glycol such as, for example, polyethyleneglycol or polypropylene glycol. In another aspect, the emulsifier ispolyethylene glycol having a molecular weight 100 Da to 5,000 Da, 200 Dato 2,500 Da, 300 Da to 1,000 Da, 400 Da to 750 Da, 550 Da to 650 Da, orabout 600 Da.

In another aspect, the emulsifier is a poloxamer. In one aspect, thepoloxamer is a nonionic triblock copolymer composed of a centralhydrophobic chain of polyoxypropylene (e.g., (poly(propylene oxide))flanked by two hydrophilic chains of polyoxyethylene (e.g.,poly(ethylene oxide)). In one aspect, poloxamer has the formula

HO(C₂H₄O)_(b)(C₃H₆O)_(a)(C₂H₄O)_(b)OH

wherein a is from 10 to 100, 20 to 80, 25 to 70, or 25 to 70, or from 50to 70; b is from 5 to 250, 10 to 225, 20 to 200, 50 to 200, 100 to 200,or 150 to 200. In another aspect, the poloxamer has a molecular weightfrom 2,000 to 15,000, 3,000 to 14,000, or 4,000 to 12,000. Poloxamersuseful herein are sold under the tradename Pluronic® manufactured byBASF. Non-limiting examples of poloxamers useful herein include, but arenot limited to, Pluronic® F68, P103, P105, P123, F127, and L121.

In another aspect, the emulsifier is composed of one or more fattyalcohols. In one aspect, the fatty alcohol is a liner or branched C₆ toC₃₅ fatty alcohol. Examples of fatty alcohols include, but are notlimited to, capryl alcohol (1-octanol), 2-ethyl hexanol, pelargonicalcohol (1-nonanol), capric alcohol (1-decanol, decyl alcohol), undecylalcohol (1-undecanol, undecanol, hendecanol), lauryl alcohol (dodecanol,1-dodecanol), tridecyl alcohol (1-tridecanol, tridecanol,isotridecanol), myristyl alcohol (1-tetradecanol), pentadecyl alcohol(1-pentadecanol, pentadecanol), cetyl alcohol (1-hexadecanol),palmitoleyl alcohol (cis-9-hexadecen-1-ol), heptadecyl alcohol(1-n-heptadecanol, heptadecanol), stearyl alcohol (1-octadecanol),isostearyl alcohol (16-methylheptadecan-1-ol), elaidyl alcohol(9E-octadecen-1-ol), oleyl alcohol (cis-9-octadecen-1-ol), linoleylalcohol (9Z, 12Z-octadecadien-1-ol), elaidolinoleyl alcohol (9E,12E-octadecadien-1-ol), linolenyl alcohol (9Z, 12Z,15Z-octadecatrien-1-ol) elaidolinolenyl alcohol (9E, 12E,15-E-octadecatrien-1-ol), ricinoleyl alcohol(12-hydroxy-9-octadecen-1-ol), nonadecyl alcohol (1-nonadecanol),arachidyl alcohol (1-eicosanol), heneicosyl alcohol (1-heneicosanol),behenyl alcohol (1-docosanol), erucyl alcohol (cis-13-docosen-1-ol),lignoceryl alcohol (1-tetracosanol), ceryl alcohol (1-hexacosanol),montanyl alcohol, cluytyl alcohol (1-octacosanol), myricyl alcohol,melissyl alcohol (1-triacontanol), geddyl alcohol (1-tetratriacontanol),or cetearyl alcohol.

In one aspect, the carrier used to produce the topical composition is amixture polyethylene and one or more fatty alcohols. For example, thecarrier is composed of 50% to 99% by weight, 75% to 99% by weight, 90%to 99% by weight, or about 95% by weight polyethylene glycol and 1% to50% by weight, 1% to 25% by weight, 1% to 10% by weight, or about 5% byweight fatty alcohol. In a further aspect, the carrier is a mixture ofpolyethylene glycol and cetyl alcohol.

The topical compositions can also include additional componentstypically present in such compositions. In one aspect, the topicalcomposition can include one or more of the following components: fats,waxes, pearlescent waxes, bodying agents, thickeners, superfattingagents, stabilizers, polymers, silicone compounds, lecithins,phospholipids, biogenic active ingredients, deodorants, antimicrobialagents, antiperspirants, swelling agents, insect repellents,hydrotropes, solubilizers, preservatives, perfume oils and dyes.Examples of each of these components are disclosed in U.S. Pat. No.8,067,044, which is incorporated by reference with respect thesecomponents.

The topical compositions composed of the micronized compositionsdescribed herein can be prepared by mixing the particles with thecarrier for a sufficient time such that the particles are evenlydispersed throughout the carrier. In the case when the carrier iscomposed of two or more components, the components can be admixed withone another prior to the addition of the micronized composition. Theamount of micronized composition present in the topical composition canvary depending upon the application. In one aspect, the micronizedcomposition is from 0.5% to 20%, 1% to 10%, 2% to 5%, or about 3% byweight of the topical composition. Exemplary procedures for makingtopical compositions described herein are provided in the Examples.

II. Applications of Micronized Compositions

The micronized compositions described herein have numerous medicalapplications. In one aspect, micronized compositions composed of amnionand intermediate tissue layer can be used as dermal fillers. The skin ismade up of three layers: the epidermis, dermis, and subcutaneous. Theepidermis is the outer layer and functions as a barrier to the externalenvironment. The dermis is the second layer of the skin containing thestructural elements, which are collagen and elastin fibers. The collagengives the skin its strength while the elastin fibers give the skin itselasticity. In between the epidermis and dermis is an area termed thedermal-epidermal junction. It interlocks forming fingerlike projections,called rete ridges, increasing the surface area of the epidermis that isexposed to the blood vessels in the dermis. The cells of the epidermisreceive its nutrients from the blood vessels in the dermis. The lastlayer of skin is the subcutaneous tissue which contains the fat cells.These fat cells make the skin look plump and youthful. It also providesinsulation to the body.

As a person ages, the skin goes through many changes that willeventually lead to wrinkles. The number of epidermal cells decreasescausing the skin to look noticeable thinner and making it more difficultfor the skin to repair itself. The dermal layer not only gets thinner,but also produces less collagen and the elastin fibers wear out causinga decrease in elasticity. These changes in the scaffolding of the skincause the skin to wrinkle and sag. The rete-ridges of thedermal-epidermal junction flatten out, making the skin more fragile andeasier for the skin to shear. The flatten rete-ridges decrease thesurface area of epidermis in contact with the dermis, which leads to adecrease in the amount of nutrients available to the epidermis. Thisalso interferes with the skin's normal repair process. In thesubcutaneous layer, the fat cells get smaller with age leading to morenoticeable wrinkles and sagging.

Amnion contains growth factors such as EGF, bFGF, and PDGF that promoteswound healing and re-epithelialization. Not wishing to be bound bytheory, the application of a topical composition composed of themicronized compositions described herein where the epithelial layer ofthe skin is disrupted can be effective in delivering the growth factorsdirectly to the injured site to promote healing. Amnion is a unique ECMdue to the presence of collagen types IV, V and VII, which enables theamnion to bind water and swell.

Similarly, the intermediate tissue layer of the amniotic membrane iscomposed largely of glycoproteins and proteoglycans, which also enablesthe intermediate tissue layer to bind water. Thus, the micronizedparticles when applied to the skin or wound help retain water in theskin, which facilitates wound healing. For example, cell migrationwithin the wound healing cascade is facilitated in a hydrophilicenvironment. The intermediate layer is also composed of collagen typesI, III, and IV. Type I collagen provides mechanical strength to skin byproviding a major biomechanical scaffold for cell attachment andanchorage of macromolecules. Type III collagen provides elasticity.Hence, by adding the intermediate tissue layer tissue to the deep dermisit will not only increase the elasticity and scaffolding of the skin, itmay make it feel softer. Another important component in the intermediatetissue layer that is beneficial to skin is proteoglycans. As discussedabove, proteoglycans allow the intermediate tissue layer to bind waterto such a large degree and swell considerably. As noted before, the fatcells in the subcutaneous layer get smaller with age leading to morenoticeable wrinkles. Thus, by injecting a dermal filler composed of themicronized compositions described herein can make the skin look morenoticeably plump and youthful.

The selection of the carrier of the topical composition can also createa hydrophilic environment in the skin, which enhances wound healing. Thesize and dimension of the microparticles can also enhance dermal woundhealing. For example, microparticles having a particle size of 20 m to100 m, or 25 μm to 75 m can be effective in dermal applications. Thus,in one aspect, the topical compositions described herein can helpprevent or reduce wrinkle formation in a subject. In another aspect, thetopical compositions can an enhance re-epithelialization of the dermalskin layer after laser-resurfacing. The topical compositions describedherein can be used alone or in combination with other skin therapiessuch as, for example, moisturizers. Vitamin (A) creams, vitamin (E),recombinant hyaluronic acid, or human, animal, and natural oils (e.g.,tee tree oil).

In other aspects, the grafts described herein can be used in orthopedicapplications (i.e., sports medicine). Sports medicine includes therepair and reconstruction of various soft-tissue injuries in or aroundjoints caused by traumas, or chronic conditions brought about byrepeated motion, in active individuals and athletes. For example, sportsmedicine includes the treatment of a variety of different injuriesassociated with, but not limited to, shoulders, elbows, feet, ankleshand and wrists. In one aspect, when the micronized composition isformulated into a liquid, it can be injected into joint capsules inorder to alleviate inflammation (e.g., tennis elbow, carpel tunnel,etc.). In other aspects, when the micronized composition is formulatedas a gel or paste, the composition can be applied to articular surfacesin order to provide medical benefits. For example, the micronizedcomposition can help reduce inflammation or swelling of an articularsurface. In other aspects, the micronized composition can help repairand/or regrow chondrocytes. In further aspects, the micronizedcompositions described herein can be used in other orthopedicapplications such as aid in the repair of periostium; help repairruptured/damaged bursa; help secure void filling material during bonerepair; or in applications involving a subject's extremities (e.g.,anti-adhesion barrier for small bone fixation, anti-adhesion barrierwhere metal plating or hardware is used, or help repair ruptured/damagedbursa).

In one aspect, the micronized compositions described herein are usefulin enhancing or improving wound healing. The types of wounds thatpresent themselves to physicians on a daily bases are diverse. Acutewounds are caused by surgical intervention, trauma and burns. Chronicwounds are wounds that are delayed in closing compared to healing in anotherwise healthy individual. Examples of chronic wound types plaguingpatients include diabetic foot ulcers, venous leg ulcers, pressureulcers, arterial ulcers, and surgical wounds that become infected.

The physician's goal when treating traumatic wounds is to heal the woundwhile allowing the patient to retain natural function in the area of thewound with minimal scaring and infection. If a wound becomes infected,it can lead to a loss of limb or life. For the most part, physiciansheal these patients without incident. However, physicians dealing withchronic wounds are mainly concerned with closing the wound as quickly aspossible to minimize the risk of an infection that could lead to loss oflimb or life. Chronic wounds are wounds on patients that havecomorbidities that complicate or delay the healing cascade. In oneaspect, the micronized compositions described herein can function as atissue regeneration template that delivers essential wound healingfactors, extracellular matrix proteins and inflammatory mediators tohelp reduce inflammation, enhance healing, and reduces scar tissueformation.

In another aspect, the micronized compositions described herein can beused to enhance wound healing and prevent scar formation as a result ofa surgical incision. In one aspect, the micronized composition can beapplied to the open incision followed by suturing the incision. Forexample, the micronized composition can be applied directly to the openincision by sprinkling the composition within the incision or byinjecting the composition into the incision using a syringe, a smallbellows device, or other related device. The micronized compositions areparticularly useful where large incisions are produced by a surgicalprocedure. An example of such a procedure involves the treatment ofspinal scoliosis, which requires a significant incision along the backof the subject. In one aspect, micronized compositions composed of anamnion/chorion laminate where the epithelium layer is intact are usefulin the healing of surgical incisions with minimal scarring. With respectto wound healing and the prevention of scar formation, the micronizedcompositions described herein can be used in combination with otherwound healing products. For example, any of the tissue grafts describedherein that are a precursor to the micronized compositions can beapplied to the wound after the micronized composition has been applied.

In another aspect, the micronized compositions described herein areuseful for addressing or alleviating complications to the spine andsurrounding regions that occur after surgery. Acute and chronic spinalinjuries and pain can be attributed to trauma and/or degenerativechanges in the spinal column. For the degenerative patient, there isusually a progression of possible surgeries depending on the patient'ssymptoms and disease state. The first surgical option when conservativetherapy has failed is a laminectomy or micro-discectomy. These minimallyinvasive procedures are intended to relieve the pain generator orstenosis of the spinal canal. If there is progression of the disease,then other surgeries may be necessary including, but not limited to, aspinal fusion. Spinal fusions may be achieved through severalapproaches; anterior (from the front through the abdomen), posterior(from the back), or lateral (through the side). Each approach hasadvantages and disadvantages. The goal is typically to remove the spinaldisc, restore disc height and fuse the two spinal vertebrae together tolimit motion and further degradation. There are also surgical optionsfor the surgeon and patient to replace the spinal disc with anartificial disc. Spine trauma is typically treated by fusing the spinelevels or if a vertebrae is crushed, the surgeon may choose to do acorpectomy and fusing across the levels that were affected.

In one aspect, the micronized compositions described herein are usefulin preventing or reducing scar formation on the spine or near the spineand sealing dural tears. Scar formation at or near the spine aftersurgery can be very debilitating and possibly require subsequentoperations to address the symptoms as discussed above. The term“anti-adhesion” is also used in the art to refer to the prevention ofscar tissue at or near the spine. In other aspects, the micronizedcompositions described herein can be used as a protective barrier, wherethe composition protects the spinal dura from post-surgical trauma fromthe surrounding surgical site. For example, the composition can preventdamage to the spinal dura caused by sharp edges from newly cut bone suchas vertebrae. In other aspects, the micronized compositions can be usedfor anterior lumbar interbody fusion, posterior lumbar interbody fusiontrans-lumbar interbody fusion, anterior cervical discectomy and fusion,micro discectomy, spinal dura repair, and as a dura sealant to preventCSF leakage.

Depending upon the surgical procedure, the micronized composition can beapplied directly to the spinal dura, the surrounding region of the spineto include nerve roots, or a combination thereof. Due to the uniquestructure of vertebrae, the micronized composition can be placed andaffixed at the appropriate position in the subject. The micronizedcompositions can also provide proximal and distal barrier coverage wherethe spinal lamina has been removed for exposure to the affected area.

The micronized compositions are useful in preventing or reducing scarformation that can result from a variety of surgical proceduresassociated with the spine. The micronized compositions can be used afterany procedure in the neck, mid-back, or lower back. Depending upon theapplication, the epithelium of the amnion can be substantially removed.For example, in posterior procedures such as a laminectomy ordiscectomy, the epithelium layer of the amnion is substantially removed.Removal of the epithelial cell layer exposes the amnion's basementmembrane layer, which increases cell signaling characteristics. This upregulation response enhances cellular migration and expression ofanti-inflammatory proteins, which inhibits fibrosis. The spinal dura istypically left unprotected following posterior procedures.

In other aspects, the epithelial cell layer of the amnion is notremoved. For example, in anterior procedures or modified anteriorprocedures such as Anterior Lumbar Interbody Fusion (ALIF) andTransforaminal Interbody Fusion (TLIF), the amnion epithelium layer isnot removed and remains intact. In these aspects, the micronizedcompositions provide additional protection to the vertebral surgicalsite by maintaining separation from the peritoneum, larger vessels, andabdominal musculature. The micronized composition serves as a reducedfriction anatomical barrier against adhesions and scaring. For example,the micronized composition can prevent scar tissue binding major bloodvessels to the spine. This is a common problem with post-spinal surgery,which requires a second surgical procedure to address this.

In other aspects, the micronized compositions formulated as a liquid,gel, or putty may be used in dental surgery to reduce inflammationrelated to gingivitis, periodontitis, mucositis, and peri-implantitis,treatment of periodontal intra-bony defects to regenerate new bone,periodontal ligament, and cementum, regenerate lost bone around dentalimplants, increase the amount of clinical attachment following osseouscontouring, treatment of gingival recession, regeneration of interdentalpapilla, either through surgical reconstruction or by directly injectingthe papilla to increase size and thickness, applied over the top of abarrier membrane or biocompatible mesh in alveolar vertical andhorizontal bone augmentations, applied over the surgical site afterprimary closure to aid in healing, applied onto autograft, xenograft,alloplast, caderivic allograft or placental allograft soft tissue graft,either before, during, or after placement of the soft tissue graft inthe treatment of gingival recession, increasing the amount of clinicalattachment, gingival augmentations around teeth and dental implants,expanding the zone of keratinized tissue, thickening overlying gingivaltissue in guided bone regeneration, mixed with a alloplast, xenograft,and or caderivic bone graft, either before, during, or after placementfor use in the treatment of intrabony defects to regenerate new bone,periodontal ligament, and cementum, in guided bone regenerationregenerate lost bone around implants, site preservation, fenestrationand dehiscence defects, primary and secondary alveolar ridgeaugmentations, sinus elevations, and gingival flap perforations. Inapplications involving dentin and pulpal tissue, reduce inflammation ofpulpal tissue, treatment of endodontic lesions, pulpal regeneration, andinjected into hollowed pulpal chamber prior to obturation in endodontictherapy. In applications involving oral mucosa tissue to reduceinflammation in oral lesions, the treatment of oral lesions, and appliedonto autograft, xenograft, alloplast, caderivic allograft or placentalallograft soft tissue graft either before, during, or after placement ofthe soft tissue graft to replace larger amounts of mucosal tissue lostthrough disease or traumatic injury.

In one aspect, the micronized compositions can be used to repairperipheral nerves. The micronized composition can be placed on arepaired nerve to prevent scar formation onto the healing nerve. Themicronized composition can also provide a protective enclosedenvironment for the repair to progress successfully. In other aspects,the micronized compositions can be manufactured into a nerveregeneration tube to guide nerve growth in a protective environmentwhere the nerve ends cannot be re-approximated. Here, nerves canre-attach up to a certain distance if the ends are allowed to meetfreely without other soft tissue interfering. In another aspect, themicronized compositions can be used to wrap nerve bundles afterprostatectomy procedures. These nerves are responsible for erectilefunction and possible continence. The micronized compositions can beapplied on the nerves to keep them from scarring and possibly damagingthe nerves.

In another aspect, the micronized compositions can be used in obstetricsand gynecological (OB/GYN) surgical procedures involving the treatmentof diseases that may be related to the fertility of the female, paincaused by the reproductive system or cancer in the reproductive system.These procedures include the removal of uterine fibroids (myomectomy),removal of ovarian cysts, tubal ligations, endometriosis treatments,removal of some cancerous or non-cancerous tumors, and vaginal slings.These procedures may be completed through a transvaginal, abdominal orlaproscopical approach.

The micronized compositions can be used as a patch to reduce the amountof scar tissue in the reproductive system after a surgical procedure.Scar tissue is another form of fibrous tissue and may also contribute tofertility problems. The ability to minimize the amount of scaring on theovaries, or within the fallopian tubes may help with post-operativefertility and even pain. In another aspect, the micronized compositionscan be used to reline the uterine wall after severe endometriosistreatments and increase the patient's ability to conceive. In a furtheraspect, the micronized compositions can be used as an anti-adhesionbarrier after removal of ovarian cyst or aid in the repair of vaginalwall erosion.

In other aspects, the micronized compositions can be used in cardiacapplications. Angina is severe chest pain due to ischemia (a lack ofblood, thus a lack of oxygen supply) of the heart muscle, generally dueto obstruction or spasm of the coronary arteries (the heart's bloodvessels). Coronary artery disease, the main cause of angina, is due toatherosclerosis of the cardiac arteries. Various open cardiac andvascular surgery procedures to remove atherosclerotic clots require therepair, reconstruction and closure of the vessel, and the support of aregenerative tissue patch to close and patch the surgical defect. Heartby-pass grafts and heart defect reconstruction (as part of an open-heartsurgical procedure) also can benefit from a patch or graft to provide abuttress to soft-tissue weakness, tissue replacement if there is a lackof suitable tissue, and also the potential to reduce adhesions to theheart itself. The micronized compositions described herein can be usedas a patch to support the repair of vascular and cardiac defects causedby operations and complications such as carotid artery repair, coronaryartery bypass grafting, congenital heart disease, heart valve repair,and vascular repair (i.e. peripheral vessels).

The micronized compositions described herein can be used in generalsurgery procedures. For example, general surgical procedures includeprocedures related to the abdominal cavity. These include theintestines, stomach, colon, liver, gallbladder, appendix, bile ducts andthyroid glands. Procedures may include hernias, polypectomy, cancerremoval, surgical treatment of Crohn's and ulcerative colitis. Theseprocedures may be done open or laparoscopically. In other aspects, themicronized compositions can be used to facilitate closure ofanastomosis, an anti-adhesion barrier for anastomosis, or ananti-adhesion barrier for hernia repair.

In other aspects, the micronized compositions can be used in ENTprocedures. Tympanoplasty is performed for the reconstruction of theeardrum (tympanic membrane) and/or the small bones of the middle ear.There are several options for treating a perforated eardrum. If theperforation is from recent trauma, many ear, nose and throat specialistswill elect to watch and see if it heals on its own. If this does notoccur or frequent re-perforation occurs in the same area, surgery may beconsidered. Tympanoplasty can be performed through the ear canal orthrough an incision behind the ear. Here, the surgeon harvests a graftfrom the tissues under the skin around the ear and uses it toreconstruct the eardrum. The micronized compositions described hereincan be used to prevent the additional trauma associated with harvestingthe patients' own tissue and save time in surgery. In other aspects, themicronized compositions can be used as a wound covering afteradenoidectomy, a wound cover after tonsillectomy, or facilitate repairof the Sniderian membrane.

In other aspects, the micronized compositions described herein can beused in plastic surgery procedures. Scar revision is surgery to improveor reduce the appearance of scars. It also restores function andcorrects skin changes (disfigurement) caused by an injury, wound, orprevious surgery. Scar tissue forms as skin heals after an injury orsurgery. The amount of scarring may be determined by the wound size,depth, and location; the person's age; heredity; and skincharacteristics including skin color (pigmentation). Surgery involvesexcision of the scar and careful closure of the defect. In one aspect,the micronized compositions described herein can be used as a patch toaid in the healing and prevention of scars; and keloid or cancerrevision/removal where careful approximation of soft-tissue edges is notachievable and scar tissue can result. Additionally, theanti-inflammatory properties of the micronized compositions can enhancehealing as well.

In other aspects, the micronized compositions can be used inophthalmological applications (e.g., on-lay grafts ocular surfacerepair) or urological applications (e.g., facilitate closure of the vasdeferens during vasectomy reversal or facilitate closure of the vasdeferens resulting from trauma).

In one aspect, the micronized compositions can be used in cranial durarepair and replacement, in the elimination of a frenum pull, theregeneration of lost patella tissue, the repair of the Schneiderianmembrane in the sinus cavity, soft tissue around dental implants,vestibuloplasty, and guided tissue regeneration.

In addition to the selection of the components used to make themicronized compositions, the size of the particles can also varydepending upon their application. In certain aspects, micronizedparticles having a larger particle size can be used in severalapplications. For example, the micronized particles (e.g., micronizedamnion/chorion tissue graft) having a particle size from 150 μm to 350μm can be effective in wound healing where it is desirable to reduce orprevent scar formation and enhance soft tissue healing. For example, themicronized particles can be injected directly into the wound. A varietyof different wounds can be treated with the micronized particles. In oneaspect, the micronized particles can be used to heal dermal wounds. Themicronized particles can be administered at any depth within the dermaltissue of a subject (e.g., subcutaneous, sub-dermal, etc.). In oneaspect, the micronized particles are useful in healing diabetic ulcers(e.g., foot ulcers). In other aspects, the dermal wounds can be trackingwounds (i.e., deep wounds that extend into the muscle tissue). Thus, thelarger micronized particles can be administered intramuscularly as voidpacking materials for deep, large wounds.

The larger particles with greater surface area are also capable ofabsorbing more fluids relative to the smaller particles. Not wishing tobe bound by theory, the larger particle size prevents or minimizes thepossibility of the particles migrating from the wound site by blood orother physiological fluids. This feature can further enhance woundhealing. The ability of the larger particles to absorb fluids makes theparticles hemostatic agents, where the particles can facilitate clottingwith blood and other physiological fluids in the wound. Thus, the largerparticles can be admixed and re-hydrated with any excipient suitable forinjection including, but not limited to, saline, blood, growth factorsolutions, and the like.

In addition to the advantages discussed above, the ability of the largermicronized particles to absorb fluids permits them to be admixed with avariety of substances (e.g., any of the bioactive agents describedherein) to produce pharmaceutical compositions with enhanced activity.For example, the larger particles can be mixed with additionalhemostatic agents to enhance blood clotting at a wound. In otheraspects, the larger particles can be admixed with autogeneous materialssuch as bone derived from the patient. Here the micronized particles canbe administered directly to the periosteal interface. In other aspects,the larger micronized particles can be admixed with fibrin glues toenhance wound healing. The micronized particles can enhance the abilityof the fibrin glue to form fibrin clots and enhance tissue repair. Thus,the larger particles in combination with the fibrin glue can furtherreduce the need of sutures typically used to close wounds.

Although the micronized compositions described herein can be applieddirectly to the tissue of a subject, they can also be applied to a wounddressing that can subsequently be applied to the subject. For example,the wound dressing can be gauze, a bandage or wrap, or any othersuitable article capable of containing or affixing the micronizedcomposition that can be applied directly to a subject.

In other aspects, the micronized compositions described herein can beapplied to a medical device such as, for example, an implantable medicaldevice. Implantable medical devices can be coated with one or moremicronized compositions described herein to provide the device withbeneficial properties when used in living tissue (e.g., enhanced woundhealing and the prevention of scaring).

Examples of suitable implantable devices that can be coated with themicronized compositions described herein include, but are not limitedto, coronary stents, peripheral stents, implants (e.g., dental,orthopedic, spinal), catheters, arterio-venous grafts, by-pass grafts,pacemaker and defibrillator leads, anastomotic clips, arterial closuredevices, patent foramen ovale closure devices, and drug deliveryballoons. The implantable device can be made of any suitablebiocompatible materials, including biostable and bioabsorbablematerials. Suitable biocompatible metallic materials include, but arenot limited to, stainless steel, tantalum, titanium alloys (includingnitinol), and cobalt alloys (including cobalt-Chromium-nickel andcobalt-chromium-tungsten alloys). Suitable nonmetallic biocompatiblematerials include, but are not limited to, polyamides, fluoropolymers,polyolefins (i.e. polypropylene, polyethylene etc.), nonabsorbablepolyesters (i.e. polyethylene terephthalate), and bioabsorbablealiphatic polyesters (i.e. homopolymers and copolymers of lactic acid,glycolic acid, lactide, glycolide, para-dioxanone, trimethylenecarbonate, epsilon-caprolactone, and the like, and combinations ofthese).

In other aspects, the micronized compositions described herein can beapplied to wound healing devices. For example, the wound healing devicecan be a bandage, wrap, gauze, suture, or any other device used to treatwound. In these aspects, the micronized composition can be coated onand/or impregnated within the device. In other aspects, the woundhealing device can be a membrane or graft used in wound healingapplications, where the micronized composition is coated on one or moresides of the membrane or graft. For example, any of the tissue graftsdescribed herein that are a precursor to the micronized compositions canbe coated with the micronized compositions.

The micronized compositions can be applied to a medical device using anytechniques known to those skilled in the art or those that may bedeveloped for applying a coating to a medical device. Examples ofsuitable techniques for applying the coating to the medical deviceinclude spraying, dip coating, roll coating, spin coating, powdercoating, and direct application by brush or needle. One skilled in theart will appreciate the many different techniques in powder coating. Themicronized compositions can be applied directly to the surface of theimplant device, or they can be applied over a primer or other coatingmaterial.

Finally, the three-dimensional constructs composed of the micronizedcompositions described herein have numerous applications. Depending uponthe application of the construct, the construct can be tailor-made forthe application. For example, if the construct is going to be used inhard bone repair or weight bearing applications, a dense construct canbe produced from micronized particles described herein with a smallerparticle sizes. Alternatively, if a softer or spongy construct is needed(e.g., a filling for a bony defect after the removal of a cyst), theconstruct can be made from micronized particles having a larger particlesize. Thus, in one aspect, the constructs described herein can be usedin orthopedic applications such as, for example, bone void filling,osteochondral repair, and articular surface repair. In another aspect,the constructs can be used in spine applications such as, for example,void filling as well as provide structural support. In dentalapplications, the constructs can be used as socket bone filler aftertooth extraction. In another aspect, the constructs may be used as ablock graft and fixed into place using bone tacks or screws in verticaland horizontal alveolar bone augmentations.

In other aspects, the three-dimensional constructs can be used incombination with any of the placental tissue described herein. Forexample, one or more placental tissues such as, for example, a layer ofamnion, chorion, or a laminate of amnion and/or chorion can be affixedto construct. In this aspect, the construct can be inserted into a woundor void, where the amnion, chorion, or laminate is on the outside of thewound and holds the construct in place, much like a bandage.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, and methods described and claimed herein aremade and evaluated, and are intended to be purely exemplary and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers (e.g., amounts, temperature, etc.) but some errors anddeviations should be accounted for. Unless indicated otherwise, partsare parts by weight, temperature is in ° C. or is at ambienttemperature, and pressure is at or near atmospheric. There are numerousvariations and combinations of reaction conditions, e.g., componentconcentrations, desired solvents, solvent mixtures, temperatures,pressures and other reaction ranges and conditions that can be used tooptimize the product purity and yield obtained from the describedprocess. Only reasonable and routine experimentation will be required tooptimize such process conditions.

Preparation of Micronized Composition Example 1

The micronized human amniotic membrane injectable was composed of humanamnion as described above and intermediate layer tissue obtained fromplacenta tissue originated in a hospital, where it is collected during aCesarean section birth. The micronization of the tissue was performedusing a Retsch Oscillating Mill MM400. Phosphate buffer was used as acarrier. The ratio of the injectable was 50 mg/mL. The concentrationratio was 60% (21 mg) amnion and 40% (14 mg) intermediate tissue layerwith 0.70 mL of phosphate buffer. The micronized composition can beadministered as a dermal filler with a 27 gauge needle in the deepdermis region. A suitable dose would be 0.5 cc to 1.0 cc of thecomposition described above.

Example 2

Amnion/chorion tissue grafts used here to produce the micronizedparticles were produced by the process described in US 2008/0046095,which is incorporated by reference in its entirety. Tissue grafts (4cm×3 cm) and two 9.5 mm steel grinding balls were placed in 50 mL vialsand the vials subsequently sealed. The vials were placed in theCryo-block, and the Cryo-block was placed in a Cryo-rack. The Cryo-rackwas placed into a liquid nitrogen holding Dewar. Tissue samples weresubjected to vapor phase cooling for no more than 30-60 minutes. TheCryo-rack was removed from the Dewar, and the Cryo-block was removedfrom the Cryo-rack. The Cryo-block was placed into the Grinder(SPEXSample Prep GenoGrinder 2010) and set at 1,500 rpm for 20 minutes. After20 minutes has elapsed, the tissue is inspected to ensure micronization.If necessary, the tissue can be placed back into the Dewar for anadditional 30-60 minutes, and moved to the grinder for an additional 20minutes to ensure sufficient micronization. Once the tissue issufficiently micronized it is sorted using a series of American StandardASTM sieves. The sieves were placed in the following order: 355 μm, 300μm, 250 μm, 150 μm, and 125 μm. The micronized material was transferredfrom the 50 mL vials to the 355 m sieve. Each sieve was agitatedindividually in order to thoroughly separate the micronized particles.Once the micronized particles have been effectively separated using thesieves, the micronized particles having particle sizes of 355 μm, 300μm, 250 μm, 150 μm, and 125 μm were collected in separate vials.

Preparation of Topical Compositions Composed of Micronized Particles

The following procedure produces a topical lotion composed of 95%polyethylene glycol (PEG) 600, 5% cetyl alcohol, and 10% water. PEG 600was poured into a beaker and warmed to 45° C. While stirring under heat,water was added to the beaker. After thoroughly mixing for five minutes,cetyl alcohol was then added while continuing to stir under heat forfive more minutes. The mixture was then allowed to cool at roomtemperature for five minutes.

Micronized amnion/chorion produced in Example 2 above (particle size 25μm to 75 μm) was then added to the mixture above to produce a 3% byweight lotion. The mixture was stirred constantly for three to fiveminutes at room temperature. The lotion was then placed into arefrigerator at 4° C. for fifteen minutes to cool. The final lotion hasa heavy cream type consistency or viscosity. When applied directly tothe skin, the lotion provides a slight moisturizing sheen with fulluptake of amnion particulate material.

Various modifications and variations can be made to the compounds,compositions and methods described herein. Other aspects of thecompounds, compositions and methods described herein will be apparentfrom consideration of the specification and practice of the compounds,compositions and methods disclosed herein. It is intended that thespecification and examples be considered as exemplary.

What is claimed:
 1. A composition comprising micronized amnion, chorion,intermediate tissue layer, or any combination thereof, wherein thecomposition is not composed solely of micronized amnion.
 2. Thecomposition of claim 1, wherein the composition comprises micronizedamnion, chorion, intermediate tissue layer, or any combination thereofas individual components.
 3. The composition of claim 1, wherein thecomposition comprises a micronized tissue graft comprising amnion,chorion, intermediate tissue layer, or any combination thereof.
 4. Thecomposition of claim 1, wherein the composition comprises micronizedamnion and intermediate tissue layer as individual components, whereinthe intermediate tissue layer has been removed from the amnion.
 5. Thecomposition of claim 4, wherein the micronized amnion has a particlesize less than 200 μm.
 6. The composition of claim 4, wherein the weightratio of amnion to intermediate tissue layer is from 10:1 to 1:10. 7.The composition of claim 4, wherein the weight ratio of amnion tointermediate tissue layer is from 2:1 to 1:1 and the micronized amnionand intermediate tissue layer has a particle size from 25 μm to 200 μm.8. The composition of claim 1, wherein the composition comprises amicronized tissue graft comprising at least two layers of chorion, atleast two layers of amnion membrane, or at least one layer of chorionand amnion membrane.
 9. The composition of claim 1, wherein thecomposition comprises a micronized amnion/chorion tissue graft.
 10. Thecomposition of claim 9, wherein the micronized particles have a particlesize less than 400 μm.
 11. The composition of claim 9, wherein themicronized particles have a particle size from 150 μm to 350 μm.
 12. Thecomposition of claim 9, wherein the weight ratio of chorion to amnion isfrom 10:1 to 1:10.
 13. The composition of claim 9, wherein the weightratio of chorion to amnion is from 4:1 to 1:1 and the micronizedparticles have a particle size from 25 μm to 200 μm.
 14. The compositionof claim 1, wherein the composition further comprises a filler.
 15. Thecomposition of claim 14, wherein the filler comprises allograftpericardium, allograft acellular dermis, Wharton's jelly, purifiedxenograft Type-1 collagen, biocellulose polymers or copolymers,biocompatible synthetic polymer or copolymer films, purified smallintestinal submucosa, bladder acellular matrix, cadaveric fascia, or anycombination thereof.
 16. The composition of claim 14, wherein the fillercomprises Wharton's jelly.
 17. The composition of claim 1, wherein theamnion, amnion, chorion, intermediate tissue layer, or any combinationthereof are cross-linked.
 18. The composition of claim 1, furthercomprising a bioactive agent.
 19. The composition of claim 18, whereinthe bioactive agent comprises a hemostatic agent, a fibrin glue, anallograft, or an autogenous material.
 20. The composition of claim 1,wherein the composition further comprises a pharmaceutically acceptablecarrier.
 21. The composition of claim 20, wherein the pharmaceuticallyacceptable carrier comprises water, aqueous hyaluronic acid, saline,Ringer's solution, dextrose solution, Hank's solution, a buffer, or anonaqueous vehicle.
 22. The composition of claim 21, wherein the buffercomprises a phosphate buffer, bicarbonate buffer, or Tris buffer. 23.The composition of claim 20, wherein the composition is injectable. 24.The composition of claim 20, wherein the composition is a liquid, gel,or paste.
 25. A method for treating or preventing wrinkles in a subject,the method comprising injecting the composition of claim 1 at the siteof an existing wrinkle in the subject or at region of the subject thatis susceptible to wrinkle formation.
 26. The use of the composition ofclaim 1 as a dermal filler.
 27. A method for enhancing wound healing,the method comprising applying the composition of claim 1 at and/or nearthe site of the wound.
 28. A method for treating or preventinginflammation in a joint of a subject, the method comprising injectingthe composition of claim 1 into the joint.
 29. A method for repairingand/or regrowing chondrocytes at an articular surface of a subject, themethod comprising applying the composition of claim 1 to the articularsurface.
 30. A method for treating or preventing inflammation at anarticular surface of a subject, the method comprising applying thecomposition of claim 1 to the articular surface.
 31. A method forpreventing or reducing scar formation on or near the spine after asurgical procedure, the method comprising applying to a subject themicronized composition of claim 1 directly to the spinal dura of thesubject or a region near the spine.
 32. The method of claim 31, whereinthe surgical procedure comprises a posterior procedure.
 33. The methodof claim 32, wherein the posterior procedure is a laminectomy ordiscectomy.
 34. The method of claim 32, wherein the posterior procedureis an Anterior Lumbar Interbody Fusion (ALIF) and TransforaminalInterbody Fusion (TLIF).
 35. The method of claim 31, wherein thesurgical procedure comprises an anterior procedure.
 36. A method fortreating a dural tear in a subject, the method comprising applyingdirectly to the dural tear the micronized composition of claim
 1. 37. Amethod for promoting the healing of a wound in a subject, the methodcomprising applying to the wound the micronized composition of claim 1,wherein the wound is in the cranial dura, a wound resulting from aperioplastic procedure, for the elimination of a frenum pull, for theregeneration of lost patella tissue, for the repair of the Schneiderianmembrane in the sinus cavity, or the soft tissue around dental implants.38. A method for promoting wound healing associated with a dentalsurgical procedure, wherein the method comprises contacting the woundwith the micronized composition of claim
 1. 39. The method of claim 38,wherein the micronized particles are used with dental implants, in thetreatment of advanced gingival recession defect, or in guided tissueregeneration.
 40. A method for promoting wound healing associated withan orthopedic application, wherein the method comprises contacting thewound with the micronized composition of claim
 1. 41. The method ofclaim 40, wherein the micronized particles are used in tendon repair,aiding in the repair of periostium, repairing ruptured/damaged bursa, orsecuring void filling materials during bone repair.
 42. A method forpromoting wound healing associated with an ENT application, wherein themethod comprises contacting the wound with the micronized composition ofclaim
 1. 43. A three-dimensional construct comprising the micronizedcomposition of claim 1, wherein the micronized particles are produced bythe process comprising (1) treating the micronized particles with across-linking agent and placing the treated particles in a mold or (2)admixing the micronized particles with an adhesive and placing theadmixture in a mold.
 44. The construct of claim 43, wherein themicronized particles comprise a micronized amnion/chorion tissue graft.45. The construct of claim 43, wherein the micronized particles comprisemicronized amnion and intermediate tissue layer.
 46. The construct ofclaim 43, wherein the adhesive comprises a fibrin sealant, acyanoacrylate, gelatin and thrombin products, polyethylene glycolpolymers, albumin, gluteraldehyde products, or any combination thereof.47. The construct of claim 43, wherein the construct further comprisesone or more placental tissues affixed to the construct.
 48. Theconstruct of claim 47, wherein the placental tissue comprises a layer ofamnion, chorion, or a laminate of amnion and/or chorion.
 49. A topicalcomposition comprising the micronized composition of claim 1 and apharmaceutically acceptable carrier.
 50. The composition of claim 49,wherein the micronized particles have a particle size from 20 μm to 100μm.
 51. The composition of claim 49, wherein the micronized particlesare from 0.5% to 20% by weight of the topical composition.
 52. Thecomposition of claim 49, wherein the micronized particles are from 2% to5% by weight of the topical composition.
 53. The composition of claim49, wherein the pharmaceutically acceptable carrier comprises asurfactant, an emulsifier, or a combination thereof.
 54. The compositionof claim 49, wherein the pharmaceutically acceptable carrier comprises apolyalkylene glycol.
 55. The composition of claim 49, wherein thepharmaceutically acceptable carrier comprises a mixture of polyalkyleneglycol and a fatty alcohol.
 56. The composition of claim 55, wherein thepolyalkylene glycol is polyethylene glycol and the fatty alcoholcomprises capryl alcohol (1-octanol), 2-ethyl hexanol, pelargonicalcohol (1-nonanol), capric alcohol (1-decanol, decyl alcohol), undecylalcohol (1-undecanol, undecanol, hendecanol), lauryl alcohol (dodecanol,1-dodecanol), tridecyl alcohol (1-tridecanol, tridecanol,isotridecanol), myristyl alcohol (1-tetradecanol), pentadecyl alcohol(1-pentadecanol, pentadecanol), cetyl alcohol (1-hexadecanol),palmitoleyl alcohol (cis-9-hexadecen-1-ol), heptadecyl alcohol(1-n-heptadecanol, heptadecanol), stearyl alcohol (1-octadecanol),isostearyl alcohol (16-methylheptadecan-1-ol), elaidyl alcohol(9E-octadecen-1-ol), oleyl alcohol (cis-9-octadecen-1-ol), linoleylalcohol (9Z, 12Z-octadecadien-1-ol), elaidolinoleyl alcohol (9E,12E-octadecadien-1-ol), linolenyl alcohol (9Z, 12Z,15Z-octadecatrien-1-ol) elaidolinolenyl alcohol (9E, 12E,15-E-octadecatrien-1-ol), ricinoleyl alcohol(12-hydroxy-9-octadecen-1-ol), nonadecyl alcohol (1-nonadecanol),arachidyl alcohol (1-eicosanol), heneicosyl alcohol (l-heneicosanol),behenyl alcohol (1-docosanol), erucyl alcohol (cis-13-docosen-1-ol),lignoceryl alcohol (1-tetracosanol), ceryl alcohol (1-hexacosanol),montanyl alcohol, cluytyl alcohol (1-octacosanol), myricyl alcohol,melissyl alcohol (1-triacontanol), geddyl alcohol (1-tetratriacontanol),cetearyl alcohol, or any combination thereof.
 57. The composition ofclaim 55, wherein the polyalkylene glycol is polyethylene glycol and thefatty alcohol is cetyl alcohol.
 58. A method for treating wrinkles in asubject, the method comprising applying the composition of claim 49 onan existing wrinkle present on subject.
 59. A method for preventingwrinkles in a subject, the method comprising applying the composition ofclaim 49 on the skin of the subject that is prone to wrinkle formation.60. A method for enhancing re-epithelialization of the dermal skinlayer, the method comprising applying the composition of claim 49 to thedamaged epithelial layer present on the skin.
 61. A method for enhancingwound healing in a subject, the method comprising injecting themicronized composition of claim 1 in or near the wound.
 62. The methodof claim 61, wherein the composition is injected sub-cutaneously,sub-dermally, intramuscularly, or at the periosteal interface.
 63. Themethod of claim 61, wherein the wound is a diabetic ulcer, a superficialskin wound, tracking wound, or a wound derived from a repeated surgicalprocedure.
 64. The method of claim 61, wherein the wound is an incisionsite for the treatment of spinal scoliosis.
 65. An implantable medicaldevice comprising the micronized composition of claim 1.